The basement membrane (BM) is a continuous structural barrier separating epithelial tissues from adjacent stroma. Major components of the BM include laminin, type I and type IV collagen, entactin, tenascin and proteoglycans, such as heparin sulfate proteoglycans. Since cells are constantly interacting with their extra-cellular environment, the BM is involved in a myriad of processes in vivo. These processes include physiological processes, such as tissue growth and development, bone remodeling, angiogenesis, reproduction, wound healing and neuron regeneration, as well as pathological processes such as tumorigenesis/metastasis (including cellular migration, invasion and angiogenesis), vascular dysfunction, arthritis and aging, and atherosclerosis. In each of these processes, cells interact with and respond to the components of the extra-cellular milieu. As a result, to study these processes in vitro the composition of the extra-cellular milieu (i.e., the BM) must be accurately and reproducibly re-created.
Taking one of the processes discussed above, tumorigenesis/metastasis, the importance of a BM system for in vitro study can be seen. Invasion of the BM is a critical stage in the complex multi-step process of metastasis. The mechanisms controlling tumor invasion through the BM involve multiple morphological and functional events which can be represented as including; (i) tumor cell adherence to the BM via specific cell surface receptors for BM components (such as, but not limited to, the laminin receptor(s); (ii) secretion of BM degrading enzymes (such as, but not limited to, collagenase); and (iii) migration of the tumor cells through BM defects into the circulatory and lymphatic systems, stimulated in part by chemotactic responses.
In order to study the mechanisms of tumor cell invasion, in vitro model systems have been developed. These include both natural and artificial BM systems. The use of naturally occurring BM systems have involved the use of intact BM such as those found in the bladder wall, amnion, lens capsule and chick chorioallantoic membranes. However, when using these naturally occurring BMs in in vitro assays, great care must be taken to ensure that the BM used is intact (i.e., there are no defects in the BM that would allow the test cell to pass through without requiring degradation of the BM). In addition, the thickness of the BM's used in various assays must be uniform. When different thicknesses of BM are used the results of invasion assays will vary dramatically. Therefore, the use of naturally occurring BM in in vitro invasiveness assays is a difficult process subject to significant inter-experiment variation. This makes the result obtained with these assays difficult to compare.
Artificial systems were developed to address these concerns. Initial artificial system utilized composite layers of extracellular matrix proteins that comprise the BM, such as purified rat fibrin, laminin and collagen. Additional artificial BM systems included reconstituted BM-matrices. Matrigel, formulated from reconstituted BM from mouse Engelbreth-Holm-Swarm tumors, is one example. The Matrigel formulation, however, was found not to mimic naturally occurring BM in composition (Matrigel essentially lacks collagen I, an essential protein required for cell-matrix interactions) and to contain significant amounts of growth factors (such as epidermal growth factor, EGF, platelet derived growth factor, PDGF, and fibroblast growth factor, FGF). Matrigel has also been reported to contain collagenase activity. Importantly, the Matrigel composition is both tumorogenic and angiogenic. In many cases, non-transformed cell types display aberrant growth characteristics and invasiveness when cultured in the presence of Matrigel. For example, endothelial cells rapidly form tube like structures when cultured in the presence of Matrigel in the absence of added growth factors or other stimulants, suggesting that growth factors and/or stimulants in the Matrigel composition stimulate this process. In addition, NG108-15 neuroblastoma plus glioma hybrid cells in culture rapidly form long neuritic processes (within 2 hours) in the absence of added growth factors or other stimulants when cultured in the presence of Matrigel, again suggesting endogenous growth factors and/or stimulants in the Matrigel formulation stimulate this process. The presence of growth factors is not surprising since Matrigel was derived from mouse tumor tissues. As a result, formulations such as Matrigel may not be suitable for the determination of invasiveness or other parameters involved in tumor cell invasion since Matrigel provides an artificially stimulated environment as a result of endogenous growth factors and/or other growth stimulants. Although the discussion above has described tumorigenesis/metastasis in detail as one application, the HuBiogel™ biomatrix described herein can also be used to address the deficiencies in other physiological and pathological models, such as those discussed above.
Modifications to the original Matrigel formulation have been attempted, including Matrigel supplemented with collagen and Matrigel containing reduced amounts of growth factors, but even these formulations have failed to provide a reliable reproducible in vitro BM system for study of a variety of physiological and pathological processes. The use of Matrigel has also been hampered by limitations due to its physical properties. For example, the physical form of Matrigel (i.e., gel or liquid) is temperature sensitive, which may impose certain limitations on its use in in vitro assays. Furthermore, it should be noted that Matrigel is derived from mouse BM components raising the possibility that using this composition for the study of human model systems may produce artificial results.
The prior art lacks a defined, biologically active biomatrix derived from human components. The instant disclosure provides such a biomatrix. The biomatrix is termed HuBiogel™.